custom monoclonal antibody development - cultek eurogentec... · † anti-peptide antibodies ......

253

Custom monoclonal antibody development

General overview 254

High quality production under ISO 9001:2000 conditions certification 254Full confidentiality guaranteed 254Specific Pathogen Free quality 254

Promoting the ethical treatment of animals 254

Mouse monoclonal 256

Rat monoclonal 257

In vitro antibody production 258

Ascites production 259

Gene immunization 261

14EUROGENTEC 2008 Catalogue > www.eurogentec.com > [email protected]

Custom monoclonal antibody development 14

Belgium• LuxembourgEurogentec Headquarters3 +32 4 372 74 005 +32 4 372 75 00u [email protected]

United KingdomEurogentec Ltd.3 +44 1794 511 4115 +44 1794 522 417u [email protected]

FranceEurogentec France s.a.s.u.3 +33 2 41 73 33 735 +33 2 41 73 10 26u [email protected]

Germany • AustriaEurogentec Deutschland GmbH3 +49 221 258 94 555 +49 221 258 94 54u [email protected]

The NetherlandsEurogentec Nederland b.v.3 +31 43 352 06 985 +31 43 354 19 65u [email protected]

SwitzerlandEurogentec s.a.succursale de Genève3 FR: +32 4 372 74 00 DE: +49 221 258 94 555 FR: +32 4 372 75 00 DE: +49 221 258 94 54

General overview

Our monoclonal antibody development services have been offered

to the scientific community since 1996. During this time we have

focused on delivering a flexible service designed to respond to your

project goals.

A dedicated project scientist handles each monoclonal antibody

development project, available for you to discuss at any time your

ongoing project goals and project progress.

All hybridomas developed at Eurogentec are the property of the

client and we guarantee not to claim any rights on the hybridoma

nor the antibodies.

Currently we offer monoclonal development in the following hosts:

† Mouse monoclonal

† Rat monoclonal

High quality production under

ISO 9001:2000 conditions

This guarantees you:

† Constant, high quality animals for immunization

† Standard procedures for immunization and blood sampling

† Excellent animal maintenance record

† Traceability

Full confidentiality guaranteed

Eurogentec guarantees full confidentiality regarding antigen

identity and project results. Feel free to contact us to put in place a

confidential disclosure agreement (CDA).

Specific Pathogen Free quality

SPF animals are tested for exposure to various viruses, bacteria,

parasites etc as recommended by FELASA. SPF animals offer

reduced background signals and higher success rates in antibody

production. Eurogentec offers SPF animals for your custom antibody

production.

Promoting the ethical treatment

of animals

Animals are maintained in an environment that is designed to minimize

their stress and exposure to danger. Animals are provided water and

feed according to their desire, are housed in groups to promote animal

interaction, and are maintained in a climate-controlled environment.

Our animal facilities comply with the highest quality standards and to

the European Community guidelines for animal housing and in vivo

experiments. Contact us for details.

3 programmes offered

† Mouse monoclonal, custom protocol

† Rat monoclonal, custom protocol

Complete range of monoclonal services

† Anti-peptide antibodies (incl. peptide synthesis)

† Anti-protein antibodies

† DNA immunization antibodies

† In vitro antibody production

† In vivo antibody production

† Antibody labelling - enzymes, biotin, fluorescence probes …

† Affinity purification

† ELISA testing

† Assay development services

Project work flow

† Comprehension of customer’s specifications

† Project plan flowchart and cost estimation

† Project approval and service agreement

† Hybridoma development with progress reports

† Delivery of hybridomas and supernatants

† Delivery of monoclonal antibody at desired scale

† Report at the end of each completed project phase

Flexibility is our service

Starting from your project goals we can provide you with a support

team for all your production and post-production related antibody

projects. Send your contact details to [email protected] and a

specialist will be pleased to discuss your project goals with you.

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255







In vitroMab production

EUROGENTEC 2008 Catalogue > www.eurogentec.com > [email protected]

Monoclonal development flowchart

Definition of Project Plan

Phase IImmunization (11 weeks)

Customer sends Antigen Eurogentec Produces Antigen

Report IELISA against antigen

Phase IIFusion (2-3 weeks)

Phase IIIScreen antigen

Screen cross-reactants(5-7 weeks)

Phase IVCloning/Subcloning

& Isotyping

In vivoMab production

Report to Customer

Report to Customer

Report to Customer

Report to Customer

Low titre

No titre

No hybridoma

No clones

Stop

Stop

Stop








Mouse monoclonalYou send us

† 250 µg of antigen per mouse (4 mouse minimum)

† May be sent lyophilized or in solution (1 ml, 250 µg/ml)

† Your ELISA protocol

You receive

† ELISA results of the polyclonal sera for mouse selection

† Fusion report – cell count of fused cells

† Screening report

- ELISA results of hybridomas against the antigen, and customer

supplied cross-reactants

- 10 ml of supernatant from up to 20 clones for customer to test

in desired application

† Cloning and isotyping report

- 10 ml of supernatant for each desired clone

† In vitro / In vivo production report

- Delivery of monoclonal antibody to desired scale

Phase I: Immunization

† A minimum of four mice (6–8 weeks old) are used for

immunization. Preimmune serum is taken on day 0.

† A total of four injections are administered (50 µg antigen per animal

per injection). The first injection on day 0 with equal parts (v/v)

of complete Freund’s adjuvant and antigen. The following three

boosts are carried out with equal parts (v/v) incomplete Freund’s

adjuvant and antigen, with three weeks between boosts.

† Selection of the best responding mouse 10 days after the last

injection using the client’s ELISA test.

Mouse monoclonal phases

Phase Reference

Phase I AS-ACMC-IM

Phase II AS-ACMC-FU08

Phase III AS-ACMC-AN08S

Phase IV AS-ACMC-CIFH

In vitro production

Mab production Reference

Roller bottle (10 mg) AS-ACMC-ROLLB

CL1000 (20-100 mg) AS-ACMC-CL1000

CP100-BR1 (100-300 mg) AS-ACMC-CP100

BR19 (> 300 mg) AS-ACMC-BR19

BR35 (> 1 g) AS-ACMC-BR35

CP2500 (> 10 g) AS-ACMC-CP2500

† If the antibody titre is too low after three booster injections, you

may request additional injections. If the ELISA report remains

negative, the immunization should be ended because the

probability of success is too low.

† If the antigen has a low immunogenicity, it can be coupled to an

appropriate protein carrier.

† Duration of this standard program: 11 weeks.

Phase II: Fusion

† The splenic lymphocytes of the best responding animal will be

fused with the cell line Sp2/O-Ag-14 using PEG (polyethylene

glycol), and the resulting hybridomas will be seeded in 96-well

plates in HAT medium. If any fusion cocktail remains, it will be

stored in liquid nitrogen.

† Duration: 2–3 weeks

Phase III: Screening and amplification of hybridomas

† IgG producing hybridomas will be screened against the target antigen

and two vials of every positive hybridoma stored in liquid nitrogen.

† The positive colonies are expanded and tested again for antibody

production with the antigen used for immunization.

† At this point, positive colonies can also be screened against

additional antigen for cross-reactivity or against a region of the

antigen of interest.

† Other screening tests can be used such as Western Blots or

immunofluorescence. Please contact us for a price offer.

† In addition, 5–10 ml supernatant of the 20 most positive

hybridomas will be sent to you for screening confirmation and

additional tests. This will ensure that the positive ELISA results

identify antibodies useful for your purpose.

† Duration: 5–7 weeks

Phase IV: Cloning and isotyping

† The specific hybridomas you have selected at the end of Phase

III will be cloned by limit dilution. The assay can be carried out as

in Phase III (screening for the antigen used for immunization and

additional screening of positive clones with additional antigens).

After cloning, three vials of every cloned hybridoma are stored

in liquid nitrogen. We send you 15–20 ml of every hybridoma

supernatant together with the frozen hybridomas.

† Subsequently, the subclass of every monoclonal antibody will

be determined by ELISA testing.

† Duration: 4 weeks

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Rat monoclonal

Phase 0 – Test phase for antigenicity† One rat is immunized in an 18-day protocol and titre is determined

by ELISA to evaluate antigenicity

† Duration 3 weeks

Phase I – Immunizations† Two rats are immunized in our 31-day protocol. Pre-immune

serum from day 0 is drawn and saved.

† 3 injections are administered

† Selection of the best responding rat for the fusion is done by

ELISA to evaluate antegenicity


Phase II – Fusion† Cells from the best responding rat are fused to rat myeloma

IR983F and a selection is made using HAT medium

† Duration 2-3 weeks

Rat monoclonal phases

Phase Reference

Test AS-RAMC-IMTEST • 1 rat

Phase I & II AS-RAMC-FU088 plates • 2 rats

Phase III AS-RAMC-AN08S • 8 plates

Phase IV AS-RAMC-CIFH • 1 clone

In vitro production

Mab production Reference

Roller bottle (10 mg) AS-RAMC-ROLLB

CL1000 (20-100 mg) AS-RAMC-CL1000

CP100-BR1 (100-300 mg) AS-RAMC-CP100

BR19 (> 300 mg) AS-RAMC-BR19

BR35 (> 1 g) AS-RAMC-BR35

CP2500 (> 10 g) AS-RAMC-CP2500

Phase III – Screening and amplification of hybridomas† Hybridomas will be screened against the target antigen and two

vials of every positive hybridoma stored in liquid nitrogen.

† The positive colonies are expanded and tested again for antibody

production with the antigen used for immunization

† At this point, positive colonies can also be screened against

additional antigens for cross-reactivy or against a region of the

antigen of interest

† Other screening tests can be used such as WB or

immunofluorescence. Please contact us for a price.

† In addition, 5-10 ml supernatant of the 20 most positive

hybridomas will be sent to you for screening confirmation and

additional test. This will ensure that the positive ELISA results

identify antibodies useful for your application

† Duration 5-7 weeks

Phase IV – Cloning and isotyping† The specific hybridomas you have selected at the end of Phase III will

be cloned by limit dilution. After cloning, three vials of every cloned

hybridomas are stored in liquid nitrogen. We send you 15-20 ml of

every hybridoma supernatant together with the frozen hybridomas.

† Subclasses of every monoclonal antibody is determined by

ELISA testing


You receive† A report at the end of each phase.

† Supernatants both before and after the cloning of the hybridomas,

in order to select the best monoclonal antibodies.

† The cloned hybridoma(s) with corresponding isotype(s).

Additional services are also offered, such as ascites purification,

antibody coupling and cell line storage. Please inquire for additional

information.

Mouse monoclonal additional service

Phase I : Immunization Reference

Per additional month and per mouse (price includes maintenance, immunization,bleedings and ELISA tests (minimum 4 mice)

AS-ACMC-IMADDM

Animal housing after mouse selection (if requested) price / month

AS-ACMC-HOUS

Phase II : Fusion Reference

Twelve 96-well plates AS-ACMC-FU12

Twenty 96-well plates AS-ACMC-FU20

Phase III : Analysis Reference

Additional screening of the positive wells. Per cross-reactant

AS-ACMC-AN08A


AS-ACMC-AN12A

Screening - 12 plates fusion AS-ACMC-AN12S

Screening with 2 antigens (used for immunization + other one)

AS-ACMC-AN20S


AS-ACMC-AN20A

Cell line storage (per year and cell line)

AS-ACMC-STOCK

Thawing - freezing (per clone)

AS-ACMC-DEFREE

Phase IV : Cloning and isotyping

Reference

Cloning and isotyping of each additional hybridoma

AS-ACMC-CIAH








In vitro antibody production

Phase I: Evaluation

Since different cell lines differ significantly in levels of antibody

production, we evaluate your hybridoma before starting the

procedure. The following parameters will be tested:

† Growth

† Mycoplasma status

† Production potential

† Immunochemical characterization

This will allow us to establish the most efficient method of growing

your hybridomas and to give you a reasonable estimate of delivery

time and price.

Phase II: Production

Antibody production will start according to the time schedule defined

with the client.

Quality Assurance

For this procedure, all reagents and culture media are obtained from

Biowhittaker-USA and its derived culture media.

We perform a rigorous Quality Control procedure with detailed product

batch characterization including HPLC, PAGE and IEF profiles.

All procedures are performed under tightly controlled conditions to

ensure a constant quality.

Different purification techniques can be used after discussion with

the client.

The client is guaranteed absolute confidentiality and security of his

cell lines and projects.

Comparison study

A one-year comparison study has been performed using different

bioreactors: culture flasks, Tecnomouse (BioIntegra), Mini-Perm

(Heraeus) and Cell Pharm System (Unisyn). Our study has clearly

shown that the most cost-effective production in the scale from

about 200 mg up to several grams of antibodies is obtained with the

highly automated Cell Pharm System (Unisyn).

Hollow-fibre technology

The Cell Pharm System makes effective use of hollow-fibre

technology. In this production method, the hybridoma cells are

placed in a bioreactor containing hollow fibres throµgh which fresh

culture medium flows. The capillaries are porous and the nutrients

can pass freely to the cells while the secreted antibodies and the

cells are excluded from the intracapillary space. Oxygen content and

medium pH are automatically regulated by a computer, ensuring that

all important parameters are controlled and available online.

In order to avoid nutrient gradients, which normally would develop,

a second pump also circulates the extracapillary medium, which

ensures a uniform nutrient distribution to all the hybridoma cells and

therefore optimal growth and production.

This system has an additional advantage in that the operator

can continually remove the secreted antibodies using the

Autoharvester, which continually samples the extracapillary medium

and automatically replaces it with fresh. The sampled antibody-

containing medium can then be processed by the purification unit

if necessary.

Closed system

As a consequence, the whole system remains closed and any risk of

contamination by bacteria or yeast is minimized. This type of reactor

can be run over months with minimal human intervention, except for

medium stock replacement.

Moreover, the system has been designed for flexibility in terms

of production quantities. Different reactor sizes are available for

optimum production of the needed quantities. This type of reactor

can easily be adapted to GMP production, as all the important

production parameters are controlled by the computer.

If you are interested in in vitro antibody production, feel free to

discuss with us all the relevant parameters of the project, (i.e. culture

conditions, antibody secretion yield in flask culture etc.), to make

sure that we can fulfill your wishes in terms of production quantities

and time frame.

Custom mouse monoclonal antibody

Phase V : In vitro production Reference

Roller bottles AS-ACMC-ROLLB

CL1000 AS-ACMC-CL1000

CP100-BR1 AS-ACMC-CP100

BR19 AS-ACMC-BR19

BR35 AS-ACMC-BR35

CP2500 AS-ACMC-CP2500

259







Ascites production

On a case by case basis where in vitro production methods fail,

Eurogentec offers ascites production.

Full confidentiality guaranteed

Antibody production can be done under the terms of a “secrecy

agreement” if requested. Eurogentec guarantees full confidentiality

regarding the hybridomas and projects.

Eurogentec will never claim any property rights on either the

hybridomas or the monoclonal antibodies. These will remain the

property of the customer.

You send us

† Completed ascites production request form.

† We need a description of the myeloma used in the fusion

process to ascertain the compatibility of the hybridoma with

Balb/c mouse. In case of non-compatibility, it is possible to

produce ascites fluid in Nude mice.

† We need approximately 2–2.5 x 106 cells per animal. A minimum

of three mice will be used per hybridoma.

† As soon as we receive your order, we will plan a date to pick up

the hybridoma at your laboratory.

We provide

† Upon arrival, the cells are washed, resuspended in a physiological

buffer and injected into pristanised Balb/c or nude mice.

† 10–15 days later, we will start the recovery of the ascites fluid

(approximately 5–10 ml per mouse).

Other information

We will keep no samples after the end of the procedure. No samples

will be transferred to any third party or be commercially used. You

will remain the sole owner of the hybridoma. Moreover, we will not

disclose to any third party that we have used your material for any

manipulation or work.

In the case where we provide ascites production with your own

hybridoma, we can not guarantee high quality ascites production if

the antibody production from your hybridoma is too low.

You receive

An average of 5–10 ml of ascites fluid per animal will be sent to

you by express courier service. Shipment costs from and to your

laboratory are included in the price per mouse as indicated in our

price list.

Custom monoclonal antibody

Ascites production # mice Reference

Balb/c* about 5 ml per animal for SP2 derived hybridomas

3 to 5 mice6 to 20 mice

› 20 mice

AS-CBAL-B

Nude* about 5 ml per animal 3 to 5 mice6 to 20 mice

› 20 mice

AS-CNUD-E

(*) depends on each hybridoma








Ascites production flowchart

Discuss with us all aspects of the project:

- small or large production scale

- Balb/c or nude mice

- additional services such as purification and labelling

For ascites production, we will choose a date to pick up the hybridoma in your laboratory.

We need approximately 5 x 106 cells per animal (minimum 3 mice per hybridoma)

Cells are washed and injected into pristanized mice. 10–15 days later, the recovery of ascites fluid will start

Affinity chromatography purification using

Protein A - Sepharose 2 weeks

Antibody labelling 2 weeks

You receive the remaining ascites fluid, affinity purified IgG and the corresponding quality control data sheet (SDS-PAGE) You will receive the labelled antibody with the corresponding Quality Control datasheet (enzymatic activity, HLPC/SDS-PAGE)

Large scale antibody production

STOP

Receive an average of 5–10 ml ascites fluid per animal

STOP

You receive the remaining ascites fluid, affinity purified IgG and the corresponding Quality Control data sheet (SDS-PAGE)

261







Gene immunization

Antibodies against your protein by direct immunization with your DNA

plasmid expression vector

How does it work ?

Plasmids harboring genes to be expressed are transferred directly to the

tissues of animal using needle injection or particle bombardment. These

plasmid vectors, containing all elements required for expression in higher

organisms then allow the in situ expression of antigenic proteins and the

immune response is induced. From this starting point, many applications

can be envisioned relating either to vaccine design and development,

basic research in immunology or polyclonal antibody production against

your protein.

What Eurogentec offers

Eurogentec has gained a wide experience in polyclonal antibody

production and has developed its own gene immunization procedure.

Simply provide us with your plasmid expression vector and we will send

you the corresponding serum at the end of each month. The animals

available for this procedure are rabbits and mice.

Uses for gene immunization

† A protein is difficult to purify or its structure is destroyed by

purification.

† Antibodies produced by genetic immunization are themselves to be

used to purify the protein.

† Antibodies need to be produced against mutated forms of the

protein.

† Proteins of poor antigenicity can be made more immunogenic by

fusing their gene to that of a highly immunogenic protein.

Immunization procedure

† In order to improve DNA uptake, we induce a cycle of muscle

degeneration and regeneration in the animal.

† The DNA is injected into the regenerating muscle. We need at least

200 µg of vector per mouse (minimum 4 mice).

† Blood sampling is performed 4 weeks later and sera are sent to you

for testing.

† You receive 10–50 µl serum per mouse (300 µl for the final bleed) or

20 ml serum per rabbit (50 ml for the final bleed).

† You decide to continue the procedure or to stop at the end of each

month according to your needs. If you continue, additional bleeds

and boosts can be performed.

† A modification of this protocol is always possible. Please contact us.

Please noteThe choice of the expression plasmid and the DNA purification procedure

are crucial. Our scientists are at your disposal to discuss this.

Gene immunization

DNA immunization Reference

4 mice/2 months AS-ADN1-MO

Additional month AS-ADN2-MO

Additional injection AS-ADN2-IN

custom monoclonal antibody development - cultek eurogentec... · † anti-peptide antibodies ......

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